Imbalances in polypeptide hormones can be important in a variety of health disorders. For example, GnRH (or LHRH) is a hormone normally found in the portal circulation between the hypothalamus and the pituitary. This 10 amino acid (AA) neuropeptide is made in the hypothalamus from a large precursor protein. Gonadotropin Releasing Hormone (GnRH) is a hormone normally found in the portal circulation between the hypothalamus and the pituitary. Pulsatile release of the decapeptide up-regulates and stimulates receptors, conversely continuous exposure down-regulates receptors. Receptors for GnRH are found on pituitary gonadotrophs where GnRH stimulates the release of luteinizing hormone (LH) and follicle stimulating hormone (FSH), hormones that are important in maintaining the reproductive function. GnRH secretion at the onset of puberty triggers sexual development, and is essential for normal sexual physiology of both males and females. In both sexes, its secretion occurs in periodic pulses usually occurring every 1-2 hours.
Circulating levels of GnRH are typically too low to measure accurately via radioimmunoassay, and quantification of peptides via traditional LC-MS/MS can be challenging. Traditionally, three methods have been used to measure peptides such as GnRH: (1) ion exchange chromatography followed by reverse phase liquid chromatography; (2) two-dimensional gel electrophoresis with excision of the peptide and further purification (e.g., liquid chromatography); and (3) antibody capture, which requires antibodies to the peptide, and subsequent purification of the peptide from the antibody. Generally, these methods are lacking in sensitivity and are too time-consuming for a high-throughput clinical assay. Thus, there is a need to develop methods to measure such peptide hormones.